Growth hormone fusion proteins

ABSTRACT

We disclose growth hormone fusion proteins that have increased in vivo stability and activity; nucleic acid molecules encoding said proteins and methods of treatment of growth hormone deficiency that use said proteins.

The invention relates to growth hormone fusion proteins; nucleic acidmolecules encoding said proteins and methods of treatment that use saidproteins.

Ligands that interact with receptors to bring about a biochemicalresponse are known as agonists and those that prevent, or hinder, abiochemical response are known as antagonists. For example, cellspecific growth factors are ligands that act as agonists and bindreceptors located at the cell surface. Activation of the receptors byligand-specific binding promotes cell proliferation via activation ofintracellular signalling cascades that result in the expression of,amongst other things, cell-cycle specific genes and the activation ofquiescent cells to proliferate.

A group of growth factors, referred to as cytokines, are involved in anumber of diverse cellular functions. These include modulation of theimmune system, regulation of energy metabolism and control of growth anddevelopment. Cytokines mediate their effects via receptors expressed atthe cell surface on target cells. Cytokine receptors can be divided intothree separate sub groups. Type 1 (growth hormone family) receptors arecharacterised by four conserved cysteine residues in the amino terminalpart of their extracellular domain and the presence of a conservedTrp-Ser-Xaa-Trp-Ser motif in the C-terminal part. The repeated Cys motifis also present in Type 2 (interferon family) and Type III (tumournecrosis factor family).

Growth hormone (GH) is an anabolic cytokine hormone important for lineargrowth in childhood and normal body composition in adults¹. The currenttherapeutic regimen for GH replacement requires once-daily subcutaneousinjections which is inconvenient and expensive. A number of approacheshave been taken to create long-acting preparations, includingpegylation² and sustained-release formulations³⁻⁵. Pegylation has thedisadvantage that it reduces affinity of hormone for receptor², andchemical modification with subsequent purification is expensive.Sustained-release formulations have proven efficacy⁴⁻⁷ but such GHpreparations are characterised by a dominant early-release profile,causing supraphysiological GH levels³, manufacture is expensive andinjections may be painful⁴. There is a need for cytokine formulationsthat minimise manufacturing costs, have good pharmacokinetic profiles,are easy to administer, and acceptable to patients.

GH acts through a cell-surface type 1 cytokine receptor (GHR). In commonwith other cytokine receptors, the extracellular domain of the GHR isproteolytically cleaved and circulates as a binding protein (GHBP)⁸.Under physiological conditions GH is in part bound in the circulation ina 1:1 molar ratio by GHBP and this complex appears to be biologicallyinactive, protected from clearance and degradation^(9,10). Across-linked complex of GH with GHBP has delayed clearance but nobiological activity¹¹. Co-administration of separately purified GHBPwith GH in a 1:1 ratio can augment the anabolic actions of GH¹². Thus,like many hormonal systems, binding in the circulation provides aninactive circulating reservoir in equilibrium with active freehormone¹³.

Cytokine hormones like growth hormone have a short plasma half-life andrequire frequent administration. For example, growth hormone (GH)replacement involves daily injections. In common with other cytokines,extracellular domain GH receptor circulates as a binding protein andnaturally prolongs GH's biological half-life.

This disclosure relates to the biological actions of a ligand-receptorfusion (LR-fusion) of GH with its extracellular domain receptor. Such agenetically engineered LR-fusion protein was purified from mammaliancell culture. In rats the LR-fusion had a 300-times reduced clearancecompared to native GH and single administration promoted growth for 10days far superior to that seen with native GH. The reduced clearance isreproducible in a primate model. The LR-fusion forms a reciprocal,head-to-tail dimer that provides a reservoir of inactive hormone asoccurs naturally with GH and its binding protein.

According to an aspect of the invention there is provided a nucleic acidmolecule comprising a nucleic acid sequence selected from:

-   -   i) a nucleic acid sequence as represented in SEQ ID NO:1;    -   ii) a nucleic acid sequence as represented in SEQ ID NO:2;    -   iii) a nucleic acid sequence as represented in SEQ ID NO:3;    -   iv) a nucleic acid sequence as represented in SEQ ID NO:4; or    -   v) a nucleic acid molecule comprising a nucleic sequence that        hybridizes under stringent hybridization conditions to SEQ ID        NO:1, SEQ ID NO: 2; SEQ ID NO: 3 or SEQ ID NO: 4, and which        encodes a polypeptide that has growth hormone receptor agonist        activity.

Hybridization of a nucleic acid molecule occurs when two complementarynucleic acid molecules undergo an amount of hydrogen bonding to eachother. The stringency of hybridization can vary according to theenvironmental conditions surrounding the nucleic acids, the nature ofthe hybridization method, and the composition and length of the nucleicacid molecules used. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed inSambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); and Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes Part I, Chapter 2(Elsevier, New York, 1993). The T_(m) is the temperature at which 50% ofa given strand of a nucleic acid molecule is hybridized to itscomplementary strand. The following is an exemplary set of hybridizationconditions and is not limiting:

Very High Stringency (Allows Sequences that Share at Least 90% Identityto Hybridize)

Hybridization:   5x SSC at 65° C. for 16 hours Wash twice:   2x SSC atroom temperature (RT) for 15 minutes each Wash twice: 0.5x SSC at 65° C.for 20 minutes eachHigh Stringency (Allows Sequences that Share at Least 80% Identity toHybridize)

Hybridization: 5x-6x SSC at 65° C.-70° C. for 16-20 hours Wash twice: 2xSSC at RT for 5-20 minutes each Wash twice: 1x SSC at 55° C.-70° C. for30 minutes eachLow Stringency (Allows Sequences that Share at Least 50% Identity toHybridize)

Hybridization: 6x SSC at RT to 55° C. for 16-20 hours Wash at leasttwice: 2x-3x SSC at RT to 55° C. for 20-30 minutes each.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleic acid sequence as represented in SEQID NO: 1.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleic acid sequence as represented in SEQID NO: 2.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleic acid sequence as represented in SEQID NO: 3.

In a preferred embodiment of the invention said nucleic acid moleculecomprises or consists of a nucleic acid sequence as represented in SEQID NO: 4.

According to an aspect of the invention there is provided a polypeptideencoded by the nucleic acid according to the invention.

According to a further aspect of the invention there is provided apolypeptide comprising an amino acid sequence selected from:

-   -   i) an amino acid sequence as represented in SEQ ID NO:5;    -   ii) an amino acid sequence as represented in SEQ ID NO:6;    -   iii) an amino acid sequence as represented in SEQ ID NO:7;    -   iv) an amino acid sequence as represented in SEQ ID NO:8;    -   v) an amino acid sequence as represented in SEQ ID NO:9;    -   vi) an amino acid sequence as represented in SEQ ID NO:10;    -   vii) an amino acid sequence as represented in SEQ ID NO:11;    -   viii) an amino acid sequence as represented in SEQ ID NO:12;        wherein said polypeptide has growth hormone receptor agonist        activity.

In a preferred embodiment of the invention said polypeptide comprises orconsists of an amino acid sequence as represented in SEQ ID NO: 5.

In a preferred embodiment of the invention said polypeptide comprises orconsists of an amino acid sequence as represented in SEQ ID NO: 6.

In a preferred embodiment of the invention said polypeptide comprises orconsists of an amino acid sequence as represented in SEQ ID NO: 7.

In a preferred embodiment of the invention said polypeptide comprises orconsists of an amino acid sequence as represented in SEQ ID NO: 8.

In a preferred embodiment of the invention said polypeptide comprises orconsists of an amino acid sequence as represented in SEQ ID NO: 9.

In a preferred embodiment of the invention said polypeptide comprises orconsists of an amino acid sequence as represented in SEQ ID NO: 10.

In a preferred embodiment of the invention said polypeptide comprises orconsists of an amino acid sequence as represented in SEQ ID NO: 11.

In a preferred embodiment of the invention said polypeptide comprises orconsists of an amino acid sequence as represented in SEQ ID NO: 12.

According to a further aspect of the invention there is provided ahomodimer comprising two polypeptides comprising or consisting of SEQ IDNO: 5.

According to a further aspect of the invention there is provided ahomodimer comprising two polypeptides comprising or consisting of SEQ IDNO: 6.

According to a further aspect of the invention there is provided ahomodimer comprising two polypeptides comprising or consisting of SEQ IDNO: 7.

According to a further aspect of the invention there is provided ahomodimer comprising two polypeptides comprising or consisting of SEQ IDNO: 8.

According to a further aspect of the invention there is provided ahomodimer comprising two polypeptides comprising or consisting of SEQ IDNO: 9.

According to a further aspect of the invention there is provided ahomodimer comprising two polypeptides comprising or consisting of SEQ IDNO: 10.

According to a further aspect of the invention there is provided ahomodimer comprising two polypeptides comprising or consisting of SEQ IDNO: 11.

According to a further aspect of the invention there is provided ahomodimer comprising two polypeptides comprising or consisting of SEQ IDNO: 12.

According to a further aspect of the invention there is provided avector comprising a nucleic acid molecule according to the invention.

In a preferred embodiment of the invention said vector is an expressionvector adapted to express the nucleic acid molecule according to theinvention.

A vector including nucleic acid (s) according to the invention need notinclude a promoter or other regulatory sequence, particularly if thevector is to be used to introduce the nucleic acid into cells forrecombination into the genome for stable transfection. Preferably thenucleic acid in the vector is operably linked to an appropriate promoteror other regulatory elements for transcription in a host cell. Thevector may be a bi-functional expression vector which functions inmultiple hosts. By “promoter” is meant a nucleotide sequence upstreamfrom the transcriptional initiation site and which contains all theregulatory regions required for transcription. Suitable promotersinclude constitutive, tissue-specific, inducible, developmental or otherpromoters for expression in eukaryotic or prokaryotic cells. “Operablylinked” means joined as part of the same nucleic acid molecule, suitablypositioned and oriented for transcription to be initiated from thepromoter. DNA operably linked to a promoter is “under transcriptionalinitiation regulation” of the promoter.

In a preferred embodiment the promoter is a constitutive, an inducibleor regulatable promoter.

According to a further aspect of the invention there is provided a celltransfected or transformed with a nucleic acid molecule or vectoraccording to the invention.

Preferably said cell is a eukaryotic cell. Alternatively said cell is aprokaryotic cell.

In a preferred embodiment of the invention said cell is selected fromthe group consisting of; a fungal cell (e.g. Pichia spp, Saccharomycesspp, Neurospora spp); insect cell (e.g. Spodoptera spp); a mammaliancell (e.g. COS cell, CHO cell); a plant cell.

According to a further aspect of the invention there is provided apharmaceutical composition comprising a polypeptide according to theinvention including an excipient or carrier.

In a preferred embodiment of the invention said pharmaceuticalcomposition is combined with a further therapeutic agent.

When administered the pharmaceutical composition of the presentinvention is administered in pharmaceutically acceptable preparations.Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents.

The pharmaceutical compositions of the invention can be administered byany conventional route, including injection. The administration andapplication may, for example, be oral, intravenous, intraperitoneal,intramuscular, intracavity, intra-articuar, subcutaneous, topical(eyes), dermal (e.g a cream lipid soluble insert into skin or mucusmembrane), transdermal, or intranasal.

Pharmaceutical compositions of the invention are administered ineffective amounts. An “effective amount” is that amount ofpharmaceuticals/compositions that alone, or together with further dosesor synergistic drugs, produces the desired response. This may involveonly slowing the progression of the disease temporarily, although morepreferably, it involves halting the progression of the diseasepermanently. This can be monitored by routine methods or can bemonitored according to diagnostic methods.

The doses of the pharmaceuticals compositions administered to a subjectcan be chosen in accordance with different parameters, in particular inaccordance with the mode of administration used and the state of thesubject (i.e. age, sex). When administered, the pharmaceuticalcompositions of the invention are applied in pharmaceutically-acceptableamounts and in pharmaceutically-acceptable compositions. When used inmedicine salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

The pharmaceutical compositions may be combined, if desired, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substancesthat are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction that wouldsubstantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier that constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation that is preferablyisotonic with the blood of the recipient. This preparation may beformulated according to known methods using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationalso may be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1, 3-butane diol.

Among the acceptable solvents that may be employed are water, Ringer'ssolution, and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono-or di-glycerides. In addition, fatty acids such as oleicacid may be used in the preparation of injectables. Carrier formulationsuitable for oral, subcutaneous, intravenous, intramuscular, etc.administrations can be found in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, PA.

According to a further aspect of the invention there is provided amethod to treat a human subject suffering from growth hormone deficiencycomprising administering an effective amount of at least one polypeptideaccording to the invention.

In a preferred method of the invention said polypeptide is administeredintravenously.

In an alternative preferred method of the invention said polypeptide isadministered subcutaneously.

In a further preferred method of the invention said polypeptide isadministered at two day intervals; preferably said polypeptide isadministered at weekly, 2 weekly or monthly intervals.

In a preferred method of the invention said growth hormone deficiency ischildhood growth hormone deficiency.

In a preferred method of the invention said growth hormone deficiency isadult growth hormone deficiency.

The treatment of growth hormone deficiency includes for example thetreatment of Turners Syndrome, Prader Willi Syndrome, Interuterinegrowth retardation, idiopathic short stature, renal failure, catabolicstates for example during chemotherapy treatment and in the treatment ofAIDS.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, means “including but not limited to”, andis not intended to (and does not) exclude other moieties, additives,components, integers or steps.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

An embodiment of the invention will now be described by example only andwith reference to the following figures:

FIG. 1 shows a schematic of relationship between GH, GH binding protein(GHBP), LR-fusion and the GHR based on published structures²⁷ (pdb3HHR):(a) The natural configuration of the GH binding to the GHBP in a1:1complex. (b) GH, released from the GHBP complex, binds to the cellsurface GH receptor. (c) The LR-fusion molecule in monomeric form withGH linked to exGHR. (d) Model for the LR-fusion forming a reciprocalhead-to-tail dimer where GH in one molecule binds to exGHR in the othermolecule. Finally in (e) the LR-fusion in monomeric form is capable ofbinding and activating the GH receptor;

FIG. 2 shows the characterisation and bioactivity of LR-fusion: (a)shows LR-fusion separated by SDS-PAGE followed by Coomassie staining(CS) and western blotting (WB), using a GH specific antibody. TheLR-fusion is approximately 75 kDa and separates into two bandsapproximately 5 kDa apart. (b) shows LR-fusion separated by native PAGEshowing that there are two protein forms fast (F) and slow (S). (c)Individual bands (F and S) from the native PAGE were excised andseparated further by SDS-PAGE under reducing conditions, followed bywestern blotting using a GH specific antibody. Both bands (F and S) runat approximately 75 kDa and separate as the doublet previouslydemonstrated. This suggests that the two distinct bands observed bynative PAGE are both composed of the 75 kDa LR-fusion and may existunder native conditions in equilibrium as a monomers and dimers. (d)Shows the elution profile for LR-fusion following gel filtration. Theseparation of 2 distinct peaks is again indicative of the presence ofthe LR-fusion as a monomer and dimer in solution. (e) Cell based GHRsignalling bioassay for GH and LR-fusion. The y-axis represents the foldinduction of corrected luciferase from a Stat 5 luciferase-reporterassay. The standard curve for GH ranges from 0, 0.25, 0.5, 1.0, 2.0 and5 nM: LR-fusion standard curve ranges from 0, 1, 2, 5, 10, 25, 50, 100and 250 nM. The maximal response for GH is achieved with 5 nM, whereasthe maximal response with the LR-fusion requires 50 to 250 nM;

FIG. 3 shows profiles of GH and LR-fusion measured after subcutaneous(sc) and intravenous (iv) administration: (a) Shows early phase (5hours) after sc administration; (b) Shows late phase (8 days) after iv;and (c) late phase after sc administration;

FIG. 4 shows the body weight change after subcutaneous treatment with OHand LR-fusion: (a) after daily GH versus placebo (vehicle only); (b)alternate day injections; (c) two injections on days 1 and 5; (d) asingle injection day 1; and (e) summary of changes in body weight afterdifferent treatment regimens. * * * =p<0.0001 GH vs LR-fusion;

FIG. 5 illustrates the in vivo activity of 1B7v0, 1B7v1, 1B7v2, and1B7v3;

FIG. 6 illustrates a time course showing body weight increase in ratsadministered 1B7v0, 1B7v1, 1B7v2, and 1B7v3;

FIG. 7 illustrates the pharmacokinetics of 1B7v0, 1B7v1, 1B7v2, and1B7v3 after subcutaneous administration;

FIG. 8 illustrates the pharmacokinetics of 1B7v0, 1B7v1, 1B7v2, and1B7v3 after intravenous administration;

FIG. 9 illustrates pharmacokinetic profiles for IB7v2 and IB7v3following sequential s/c dosing of 1 mg/kg in rhesus monkey. Red dottedline illustrates the minimum effective concentration for human growthhormone.

Table 1 shows results (mean±sem) after 10 days treatment with GH orLR-fusion in hypophysectomised rats.

Materials and Methods

Use of animals and human samples. The use of human samples was approvedby the local ethics committee and patients gave informed consent. Allthe experiments have been conducted in compliance with the French laws(Council Directive N° 86/609/EEC of 24 Nov. 1986) relating to theprotection of animals used for experimental or other scientific purpose.

Materials. All the materials were purchased from Sigma (Poole, UK)unless otherwise stated. Recombinant GH was purchased from Pfizer,recombinant E. coli derived human GH binding protein used in bindingassays was a gift from DSL (DSL Research Reagents, Oxfordshire, UK), andiodinated GH a gift from NovoNordisk (NovoNordisk Park, Denmark). GH andGHR mAbs used for purification and characterisation were in-housematerials (CS) except mAbs B07b and B24a which were a gift from Dr.Skriver (NovoNordisk Park, Denmark) and mAb 263 (AbD Serotec,Kidlington, Oxford, UK).

Purification of GH-exGHR LR-fusions. Human GH and GH receptor wereamplified by RT-PCR from human pituitary and liver respectively andcloned into the vector, pSecTag-V5/FRT/Hist-TOPO (Invitrogen, Paisley,UK) under the human GH secretion signal sequence. Four repeats of aGly₄Ser linker were used to link the native C-terminus of human GH tothe native N-terminus of the human GHR. Stable clones were made in CHOFlp-In cells (Invitrogen, Paisley, UK), adapted to protein free mediaand grown in suspension culture. LR-fusion expression was confirmed byan in-house ELISA. Affinity purification was performed using a GH mAbcolumn.

Transcription bioassays. These were performed as previously described inhuman 293 cells stably expressing the human GHR¹⁶.

ELISA. An in house GH and LR-fusion ELISA has been established based onthe sandwich ELISA format. In the assay, standards (GH or LR-fusion),controls and unknowns are incubated with biotin-labelled mouse antibodyto human GH (mAb 7F8) in wells pre-coated with a mouse antibody to humanGH antibody (mAb 10A7). The detection limit for the assay is 2.5 pg andthe intra and inter assay CV is <10%. The IGF-I ELISA was purchased fromDSL (DSL-10-2900 ACTIVE mouse/rat IGF-I kit; DSL Research Reagents,Oxfordshire, UK).

Pharmacokinetic studies. Seven weeks old normal Sprague Dawley rats fromJanvier (Le Genest Saint Isle, France) were used for pharmacokineticstudies. Sc or iv administration (penile vein) and blood withdrawal(orbital sinus) were conducted under isoflurane anaesthesia. The rats(n=4-6/group) were injected iv or sc with of 0.1 mg/kg rhGH orLR-fusion. Blood samples were collected from the retro-orbital plexus.Serum was harvested and stored at −70° C. until assays. Pharmacokineticparameters were estimated by fitting values of hormone concentrationversus time to compartmental models using non-linear least-squaresregression analysis. Clearance values were normalized to animal weight.Clearance rate per animal weight and terminal half lives (t_(1/2)) werecalculated using the coefficient and exponents obtained from the ivbolus model fits.

Primate Pharmacokinetic Study

The test substances IB7v2 and IB7v3 were formulated in solutionscontaining 11.9 mM sodium and potassium phosphates, 137 mM sodiumchloride, 2.7 mM potassium chloride, 0.01% polysorbate 80; pH of thesolution was adjusted to 7.4.

Study Design

The animals were assigned to 4 treatment groups (1 vehicle, 1 IB7v2 testgroup, 1 IB7v3 test group), comprising 3 males in the vehicle group and4 males per group in the 2 treatment groups. The dose levels and volumesadministered were as outlined in the table below:

# Monkeys Dose Group Treatment (male) (mg/kg/dose) Dose Volume 1 Vehicle3 0 0.2 mL/kg, on (control) days 1, 15 2 IB7v2 4 1 0.2 mL/kg, on days 1,15 3 IB7v3 4 1 0.2 mL/kg, on days 1, 15

Blood samples were obtained from all animals throughout the study inorder to determine the concentration of the appropriate test material inserum. These samples were taken at a number of time points throughoutthe study.

Clinical Endpoints and Measurements

The serum concentration of IB7v2 and IB7v3 was determined using avalidated ELISA method. The pharmacokinetic profile for each of theprotein was determined by plotting the concentration for each of theprotein in serum versus time using WinNonlin Pro (v4.0.1) software.

Growth studies. The growth studies used hypophysectomized rats and wereperformed on Sprague Dawley rats from Charles River Laboratories(Larbresle, France). Rats were hypophysectomized under isofluraneanaesthesia at 4 weeks of age by the breeder and delivered one weekafter selection on body weight criteria for successful surgery. Animalswere individually caged and allowed another week of rest before enteringthe experimental phase. The injection solutions of excipient, rhGH andLR-fusion never exceeded 2 ml/kg. The rats were weighed daily anddepending on the administration protocol, received injections of thetest substances for 10 days.

Characterisation of LR-fusions. Conformation of the LR-fusion wasexamined using a panel of 16 conformationally sensitive hGH receptormAbs. Denaturing, native gels and western blotting were used to analysethe LR-fusion and western blotting performed with non-conformationallysensitive to GH. The form of the LR-fusion protein in solution wasdefined by gel filtration using a Superose G200 analytical column andanalytical ultracentrifugation. Analytical ultracentrifugation (AUC) wasperformed by sedimentation velocity (Analytical service, Dr Andy Barry,Astbury, Leeds University, Leeds, UK).

Statistics. Two groups were compared with a Student's test if theirvariance was normally distributed or by a Student-Satterthwaite's testif not normally distributed.

Distribution was tested with an F test. One-way ANOVA was used tocompare the means of 3 or more groups and if the level of significancewas p<0.05 individual comparisons were performed with Dunnett's tests.All statistical tests were two-sided at the 5% level of significance andno imputation was made for missing values.

EXAMPLES Design and Characterisation of LR-Fusion

A recombinant gene encoding human GH linked to the A & B domains of theGHR extracellular domain (exGHR1-238) via a flexible (Gly₄Ser)₄ linker,was generated (FIG. 1 c). This LR-fusion was expressed in CHO cells andpurified using mAb antibody to GH affinity media to >95% purity (FIG. 2a). The LR-fusion was screened by ELISA using 16conformationally-sensitive mAbs. All these mAbs bound the LR-fusion withaffinity comparable to that for GHBP derived from human serum. Coomassiestaining and western blotting of SDS-PAGE gels showed the LR-fusionprotein to separate as a doublet of approximately 75 kDa with anapproximate 5 kDa difference between the two bands. Native PAGE gelanalysis (FIG. 2 b) showed no evidence of aggregation. The LR-fusionappeared as two distinct forms. These distinct protein forms, fast (F)and slow (S), were excised from the native PAGE gel and then re-analysedby SDS-PAGE under reducing conditions. Both F & S forms from the nativePAGE consisted of the 75 kDa doublet (FIG. 2 c). The evidence for theexistence of two forms of LR-fusion in solution was confirmed byanalytical gel filtration (FIG. 2 d). These results are consistent withthe LR-fusion existing as a dimer in solution. This was confirmed byanalytical ultracentrifugation where the size of the monomer wasconfirmed at 75 kDa.

In Vitro Bioassay and Pharmacokinetics

The in vitro bioactivity of the LR-fusion was tested using a GH-specificluciferase reporter assay¹⁶. The LR-fusion had approximately 10% of thebioactivity compared to GH in this static assay system, although theLR-fusion was capable of stimulating maximal response albeit at a higherconcentration than GH (FIG. 2 e). The LR-fusion's pharmacokineticprofile was examined in normal rats after single subcutaneous (sc) orintravenous (iv) injection (FIG. 3). The LR-fusion demonstrated delayedclearance irrespective of the route of administration and delayedabsorption after sc administration. After an iv bolus the terminalhalf-life of the LR-fusion was 21±2 h, and clearance 3.3±0.9 Theclearance of the LR-fusion was 300-times slower than GH^(2,12). Aftersingle sc administration the LR-fusion had a delayed peak compared to GH(30 vs 1 hour). The LR-fusion was still detectable at 8 days whilst GHwas undetectable at 6 hours. We examined whether the exceptionalpharmacokinetics of the LR-fusion were related to size. Two variantLR-fusion molecules with identical linkers were tested: one an LR-fusionof GH to only the B domain exGHR (55 kDa) and the other a tandem (GHlinked to GH) linked to exGHR (100 kDa). Both the 55 kDa and 100 kDaproteins showed increased agonist activity in the bioassay compared tothe original 75 kDa LR-fusion but for both the circulating half-life was<4 hours after iv administration (precise half-life determination wasnot possible as the sampling protocol used expected a longer half-life).The results confirm that the exceptional pharmacokinetics of theoriginal 75 kDa LR-fusion was not solely related to molecular weight.

The pharmacokinetic profiles in the primate study for IB7v2 and IB7v3following two sequential s/c doses of 1 mg/kg at t-0 and t-336 hours(14-days) are shown in FIG. 9. It can be noted that the levels reach amaximum rapidly and declines over a significantly extended period oftime as compared to native human growth human which has a half-life of<1-hour. The levels are also maintained well above the minimum effectiveconcentration, as defined by the dotted line over the duration of thesequential dosing.

Superiority of LR-Fusion Growth Promotion over GH

To test biological activity, the LR-fusion and GH were administered tohypophysectomised (GH-deficient) rats. Daily administration of GHinduced continuous growth over 10 days. The LR-fusion was then comparedto GH with either alternate day sc injections, or two injections over 10days, or a single injection. For all experiments equimolar doses of GHand LR-fusion were used with the same total dose being given over the 10day period: 220 μg/kg/day, approximately 10 nmol over 10 days similar tothe dose previously used to obtain a maximal growth response¹². TheLR-fusion promoted an increase in weight gain which was greater than GHunder the same injection protocol and similar to that seen after dailyGH injections (FIG. 4 and Table). GH appeared only to promote weightgain in the 24 hours post injection. In contrast, the LR-fusion producedcontinuous weight gain over 10 days even when given as a singleinjection. A similar pattern of growth was seen in femur, tibia, thymus,liver and kidney (Table). The 10-day terminal bleed from all animals wasanalysed for the GH-dependent biomarker, insulin-like growth factor-I(IGF-I), and GH and LR-fusion levels (Table). IGF-I levels weresignificantly elevated after LR-fusion administration even after singleinjection and were significantly greater than those seen after dailyinjection of GH. Levels of GH were undetectable in the terminal bleedafter all injection regimens whereas LR-fusion levels were detectable 10days after a single injection.

We have demonstrated that an LR-fusion of GH generates a potent agonist.We propose that the ability of the molecule to form a head-to-tailreciprocal dimers (FIG. 1 d) is responsible for its enhanced in vivobioactivity.

The design of the LR-fusion was based on the known crystal structure ofthe GHR¹⁷. We used a flexible Gly₄Ser linker with 4 repeats (predictedlength of 80 Å). This long linker was chosen as a relatively flexibletether between GH and the GHR such that the GH moiety could stillinteract with the cell surface GHR (FIG. 1 e). Similar Gly₄Ser linkershave been used in recombinant single chain Fv antibody productionbecause of stability and lack of immunogenicity¹⁸.

The LR-fusion was appropriately folded, appearing on both native PAGEgels and in gel filtration as two distinct species, i.e. potentiallymonomer and dimer. The presence of dimers was confirmed by analyticalultracentrifugation. We propose that the LR-fusion forms a reciprocalhead-to-tail dimer through intermolecular binding of the GH moiety ineach LR-fusion molecule to the receptor moiety in the other (FIG. 1 d).The LR-fusion appeared as two bands on SDS-PAGE, with a molecular weightdifference of 5 kDa, presumably due to glycosylation^(19,20).

The LR-fusion was more potent in vivo compared to GH but in vitrobioactivity was 10-times less. This discrepancy can be attributed todimerisation of the LR-fusion. In a static in vitro bioassay the dimerwould be biologically inactive as seen with the native GH/GHBPcomplex^(21,22). However, in vivo the dimer provides a reservoir ofinactive hormone in equilibrium with biologically active monomer.

After iv administration to rats our LR-fusion had a 300-times reducedclearance compared to GH and a 10 to 30-times reduced clearance comparedto that previously reported for a GH/GHBP complex or conjugate^(11,12).We tested two other LR-fusion variants one of 55 kDa and the other100kDa. Neither protein showed the same delayed clearance. We thereforeconclude that it is not monomeric size alone that is responsible forLR-fusion delayed clearance. The renal contribution to GH clearance hasbeen estimated to be 25-53% in humans²³ and 67% in rats²⁴. Thereforereducing renal clearance alone can only be predicted to approximatelyhalve GH clearance². As GH clearance is relatively independent of a GHreceptor mechanism²⁵ it is presumed that proteolysis is a majorcontributor. We propose that the greatly reduced clearance of ourLR-fusion is attributable to both reduced renal clearance and aconformation that prevents proteolysis.

In hypophysectomised rats our LR-fusion given only once during 10 daysproduced a similar increase in weight to that seen with daily injectionsof GH. It has previously been shown that GHBP co-administered as 1:1molar complex with GH augments growth¹². Using the same protocol ourLR-fusion protein promoted growth over 10 days after a single injectionwhereas the GH/GHBP complex required daily injections and our LR-fusiongenerated a higher IGF-I level than that seen after GH/GHBPco-administration. GH is biologically inactive when conjugated to GHBPand the non-covalently linked complex lacks the stability of theLR-fusion^(11,12). The greater biological action of the LR-fusion mayrelate to its increased stability and its ability to activate the GHR inmonomeric form.

In humans IGF-I levels are generally a good biomarker of GH activity.However, in hypophysectomised rats IGF-I levels do not always reflectthe growth response to GH^(2,12.) LR-fusion administration resulted inclearly elevated IGF-I levels compared to GH injection. We suggest thatthe dose-response to GH of growth and IGF-I differs in hypophysectomisedrats. Thus, the dose of LR-fusion used in our study was in excess ofthat required to promote a maximal growth response, but still capable ofstimulating IGF-I generation. Rats display more rapid renal clearancethan humans making it difficult to predict the dosing regimen that willbe required in man. One might expect that the LR-fusion could be used atlower doses and much less frequently than GH.

Fusions of cytokine hormones with serum albumin and pegylation have beenused to prolong circulating half-life^(2,26). Our LR-fusion molecule hasmajor advantages over these two approaches. Pegylation is highlyeffective at delaying the clearance of proteins, but requires chemicalmodification and reduces the affinity of ligand for its receptor². Thus,with pegylation a greater dose is required whereas with our LR-fusion asimilar dose has a greater effect than native GH. Regarding the GHfusion with albumen, Albutropin, relatively little is known as it isunderstood that this was withdrawn from clinical studies. In the one PKstudy²⁶ Albutropin had 6-times longer terminal half-life when givens.c.compared to GH whereas our LR-fusion protein has a 100-times longerterminal half-life given i.v. compared to that published for GH¹² (Fornative human GH: clearance value of 18.6 ml/min.kg=1116 ml/hr.kg andVd=336 ml/kg thus T1/2=0.693×336/1116=0.21 hrs.). GH naturally binds tocirculating exGHR and therefore our LR-fusion is unlikely to beimmunogenic compared to fusions with other proteins and extensive insilico T cell epitope screening showed no sites in the LR-fusionmolecule (data not shown).

REFERENCES

-   1. Woodhouse, L. J., Mukherjee, A., Shalet, S. M. & Ezzat, S. The    influence of growth hormone status on physical impairments,    functional limitations, and health-related quality of life in    adults. Endocr Rev. 27, 287317 (2006).-   2. Clark, R. et al. Long-acting growth hormones produced by    conjugation with polyethylene glycol. Journal of Biological    Chemistry. 271, 2196921977 (1996).-   3. Cook, D. M. et al. The pharmacokinetic and pharmacodynamic    characteristics of a long-acting growth hormone (GH) preparation    (nutropin depot) in GH-deficient adults. J Clin Endocrinol Metab.    87, 450814 (2002).-   4. Reiter, E. O. et al. A multicenter study of the efficacy and    safety of sustained release GH in the treatment of naive pediatric    patients with GH deficiency. J Clin Endocrinol Metab. 86, 47006    (2001).-   5. Jostel, A., Mukherjee, A., Alenfall, J., Smethurst, L. &    Shalet, S. M. A new sustained-release preparation of human growth    hormone and its pharmacokinetic, pharmacodynamic and safety profile.    Clin Endocrinol (Oxf). 62, 6237 (2005).-   6. Laursen, T. et al. Long-term effects of continuous subcutaneous    infusion versus daily subcutaneous injections of growth hormone (GH)    on the insulin-like growth factor system, insulin sensitivity, body    composition, and bone and lipoprotein metabolism in GH-deficient    adults. J Clin Endocrinol Metab. 86, 12228 (2001).-   7. Laursen, T., Jorgensen, J. O., Jakobsen, G., Hansen, B. L. &    Christiansen, J. S. Continuous infusion versus daily injections of    growth hormone (GH) for 4 weeks in GH-deficient patients. J Clin    Endocrinol Metab. 80, 24108 (1995).-   8. Muller-Newen, G., Kohne, C. & Heinrich, P. C. Soluble receptors    for cytokines and growth factors. [Review] [58 refs]. International    Archives of Allergy & Immunology. 111, 99106 (1996).-   9. Baumann, G., Amburn, K. D. & Buchanan, T. A. The effect of    circulating growth hormone-binding protein on metabolic clearance,    distribution, and degradation of human growth hormone. J Clin    Endocrinol Metab. 64, 65760 (1987).-   10. Baumann, G. Growth hormone heterogeneity: genes, isohormones,    variants, and binding proteins. Endocrine Reviews 12, 424449 (1991).-   11. Baumann, G., Shaw, M. A. & Buchanan, T. A. In vivo kinetics of a    covalent growth hormone-binding protein complex. Metabolism. 38,    3303 (1989).-   12. Clark, R. G. et al. Recombinant human growth hormone    (GH)-binding protein enhances the growth-promoting activity of human    GH in the rat. Endocrinology. 137, 43084315 (1996).-   13. Baumann, G. Growth hormone binding protein-errant receptor or    active player? [editorial]. Endocrinology. 136, 377378 (1995).-   14. Ayling, R. M. et al. A dominant-negative mutation of the growth    hormone receptor causes familial short stature. Nature Genetics. 16,    1314 (1997).-   15. Ross, R. J. et al. A short isoform of the human growth hormone    receptor functions as a dominant negative inhibitor of the    full-length receptor and generates large amounts of binding protein.    Molecular Endocrinology. 11, 265273 (1997).-   16. Ross, R. J. M. et al. Binding and functional studies with the    growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal    effects of pegylation and evidence that it binds to a receptor    dimer. Journal of Clinical Endocrinology & Metabolism. 86, 17161723    (2001).-   17. Cunningham, B. C. et al. Dimerization of the extracellular    domain of the human growth hormone receptor by a single hormone    molecule. Science. 254, 821825 (1991).-   18. Huston, J. S., Tai, M. S., McCartney, J., Keck, P. &    Oppermann, H. Antigen recognition and targeted delivery by the    single-chain Fv. Cell Biophys. 22, 189-224 (1993).-   19. Herington, A. C., Smith, A. I., Wallace, C. & Stevenson, J. L.    Partial purification from human serum of a specific binding protein    for human growth hormone. Mol Cell Endocrinol. 53, 2039 (1987).-   20. Frick, G. P., Tai, L. R., Baumbach, W. R. & Goodman, H. M.    Tissue distribution, turnover, and glycosylation of the long and    short growth hormone receptor isoforms in rat tissues.    Endocrinology. 139, 282430 (1998).-   21. Mannor, D. A., Winer, L. M., Shaw, M. A. & Baumann, G. Plasma    growth hormone (GH)-binding proteins: effect on GH binding to    receptors and GH action. J Clin Endocrinol Metab. 73, 304 (1991).-   22. Lim, L., Spencer, S. A., McKay, P. & Waters, M. J. Regulation of    growth hormone (GH) bioactivity by a recombinant human GH-binding    protein. Endocrinology. 127, 128791 (1990).-   23. Haffner, D., Schaefer, F., Girard, J., Ritz, E. & Mehls, O.    Metabolic clearance of recombinant human growth hormone in health    and chronic renal failure. J Clin Invest. 93, 116371 (1994).-   24. Johnson, V. & Maack, T. Renal extraction, filtration,    absorption, and catabolism of growth hormone. American Journal of    Physiology 233, F185F196 (1977).-   25. Veldhuis, J. D. et al. Impact of experimental blockade of    peripheral growth hormone (GH) receptors on the kinetics of    endogenous and exogenous GH removal in healthy women and men.    Journal of Clinical Endocrinology & Metabolism 87, 57375745 (2002).-   26. Osborn, B. L. et al. Albutropin: a growth hormone-albumin fusion    with improved pharmacokinetics and pharmacodynamics in rats and    monkeys. Eur J Pharmacol 456, 14958 (2002).-   27. de Vos, A. M., Ultsch, M. & Kossiakoff, A. A. Human growth    hormone and extracellular domain of its receptor: crystal structure    of the complex. Science 255, 306312 (1992)

TABLE 1 Variable at 10 t-test p days Placebo Treatment GH LR-fusion GHvs LR Weight 86.3 ± 1.6  Daily injections 103.3 ± 1.4  na na (g)Injections every 2 days 95.9 ± 0.8  102.2 ± 1.6  <0.0001 Injectionsevery 5 days 88.4 ± 2.1  101.1 ± 0.7  <0.0001 Single injection 93.2 ±2.9  108.3 ± 1.5  <0.0001 Change in 1.43 ± 0.96 Daily injections 16.4 ±0.8  na na weight from Injections every 2 days 9.9 ± 0.5  17 ± 1.50.0003 baseline Injections every 5 days 4.5 ± 1.3 14.8 ± 0.9  <0.0001(g) Single injection 5.0 ± 0.1 17.2 ± 1.1  <0.0001 Change in 0.00 ± 0.25Daily injections 0.83 ± 0.26 na na Femur Length Injections every 2 days0.99 ± 0.18 1.08 ± 0.07 0.667 (mM) Injections every 5 days 0.44 ± 0.211.29 ± 0.22 0.0194 Single injection na na na Change in 0.00 ± 0.02 Dailyinjections 0.03 ± 0.01 na na Tibia weight Injections every 2 days 0.06 ±0.02 0.05 ± 0.01 0.52 (g) Injections every 5 days 0.01 ± 0.01 0.07 ±0.02 0.027 Single injection na na na Change in 0.00 ± 21   Dailyinjections 79 ± 20 na na Thymus weight Injections every 2 days 43 ± 6 142 ± 22  0.0054 (mg) Injections every 5 days 35 ± 12 120 ± 15  0.0132Single injection −13 ± 22   117 ± 21  0.0017 Change in  0 ± 167 Dailyinjections 123 ± 170 na na Liver weight Injections every 2 days 362 ±74  587 ± 206 0.056 (mg) Injections every 5 days 402 ± 236 407 ± 1160.073 Single injection na na na Change in  0 ± 11 Daily injections 51 ±22 na na Kidney weight Injections every 2 days 45 ± 26 75 ± 21 0.0053(mg) Injections every 5 days  5 ± 26 67 ± 12 0.0273 Single injection  7± 22 78 ± 15 0.0062 IGF-I 51 ± 12 Daily injections 92 ± 30 na na(ng.ml⁻¹) Injections every 2 days 92 ± 30 329 ± 35  0.0005 Injectionsevery 5 days 55 ± 15 205 ± 5  <0.0001 Single injection  18 ± 2.5 198 ±66  0.0146 GH or Chimera nd Daily injections nd na na by ELISAInjections every 2 days nd 44 ± 15 0.015 (nM) Injections every 5 days nd23 ± 5  0.0015 Single injection nd 3.2 ± 1.2 0.0193 nd = Not Detectablena = Not Analyzed

1. A nucleic acid molecule comprising a nucleic acid sequence selectedfrom the group consisting of: i) the nucleic acid sequence asrepresented in SEQ ID NO:1; ii) the nucleic acid sequence as representedin SEQ ID NO:2; iii) the nucleic acid sequence as represented in SEQ IDNO:3; iv) the nucleic acid sequence as represented in SEQ ID NO:4; andv) a nucleic acid molecule comprising a nucleic sequence that hybridizesunder stringent hybridization conditions to the nucleic acid sequence asrepresented in SEQ ID NO:1, SEQ ID NO: 2; SEQ ID NO: 3 or SEQ ID NO: 4,and which encodes a polypeptide that has growth hormone receptor agonistactivity.
 2. The nucleic acid molecule according to claim 1, comprisingthe nucleic acid sequence as represented in SEQ ID NO:
 1. 3. The nucleicacid molecule according to claim 1, comprising the nucleic acid sequenceas represented in SEQ ID NO:
 2. 4. The nucleic acid molecule accordingto claim 1, comprising the nucleic acid sequence as represented in SEQID NO:
 3. 5. The nucleic acid molecule according to claim I, comprisingthe nucleic acid sequence as represented in SEQ ID NO:
 4. 6. Apolypeptide encoded by the nucleic acid molecule according to claim 1.7. A polypeptide comprising an amino acid sequence selected from thegroup consisting of: i) the amino acid sequence as represented in SEQ IDNO:5; ii) the amino acid sequence as represented in SEQ ID NO:6; iii)the amino acid sequence as represented in SEQ ID NO:7; iv) the aminoacid sequence as represented in SEQ ID NO:8; v) the amino acid sequenceas represented in SEQ ID NO:9; vi) the amino acid sequence asrepresented in SEQ ID NO:10; vii) the amino acid sequence as representedin SEQ ID NO:11; and viii) the amino acid sequence as represented in SEQID NO:12; wherein said polypeptide has growth hormone receptor agonistactivity.
 8. The polypeptide according to claim 7, wherein saidpolypeptide comprises the amino acid sequence as represented in SEQ IDNO:
 5. 9. The polypeptide according to claim 7, wherein said polypeptidecomprises the amino acid sequence as represented in SEQ ID NO:
 6. 10.The polypeptide according to claim 7, wherein said polypeptide comprisesthe amino acid sequence as represented in SEQ ID NO:
 7. 11. Thepolypeptide according to claim 7, wherein said polypeptide comprises theamino acid sequence as represented in SEQ ID NO:
 8. 12. A polypeptideaccording to claim 7, wherein said polypeptide comprises the amino acidsequence as represented in SEQ ID NO:
 9. 13. The polypeptide accordingto claim 7, wherein said polypeptide comprises the amino acid sequenceas represented in SEQ ID NO:
 10. 14. The polypeptide according to claim7, wherein said polypeptide comprises the amino acid sequence asrepresented in SEQ ID NO:
 11. 15. The polypeptide according to claim 7,wherein said polypeptide comprises the amino acid sequence asrepresented in SEQ ID NO:
 12. 16. A homodimer comprising twopolypeptides according to claim 7, each of the two polypeptidescomprising the amino acid sequence represented in SEQ ID NO:
 5. 17. Ahomodimer comprising two polypeptides according to claim 7, each of thetwo polypeptides comprising the amino acid sequence represented in SEQID NO:
 6. 18. A homodimer comprising two polypeptides according to claim7, each of the two polypeptides comprising the amino acid sequencerepresented in SEQ ID NO:
 7. 19. A homodimer comprising two polypeptidesaccording to claim 7, each of the two polypeptides comprising the aminoacid sequence represented in SEQ ID NO:
 8. 20. A homodimer comprisingtwo polypeptides according to claim 7, each of the two polypeptidescomprising the amino acid sequence represented in SEQ ID NO:
 9. 21. Ahomodimer comprising two polypeptides according to claim 7, each of thetwo polypeptides comprising the amino acid sequence represented in SEQID NO:
 10. 22. A homodimer comprising two polypeptides according toclaim 7, each of the two polypeptides comprising the amino acid sequencerepresented in SEQ ID NO:
 11. 23. A homodimer comprising twopolypeptides according to claim 7, each of the two polypeptidescomprising the amino acid sequence represented in SEQ ID NO:
 12. 24. Avector comprising a nucleic acid molecule according to claim
 1. 25. Avector according to claim 24 wherein said vector is an expressionvector.
 26. An isolated host cell transfected or transformed with avector according to claim
 25. 27. An isolated host cell according toclaim 26 wherein said cell is a eukaryotic cell.
 28. An isolated cellaccording to claim 26 wherein said cell is a prokaryotic cell.
 29. Apharmaceutical composition comprising a polypeptide according to claim7, and an excipient or carrier.
 30. A method to treat a human subjectsuffering from growth hormone deficiency, comprising: administering aneffective amount of at least one polypeptide according to claim 7,thereby treating the subject suffering from the growth hormonedeficiency.
 31. The method according to claim 30 wherein saidpolypeptide is administered intravenously.
 32. The method according toclaim 30 wherein said polypeptide is administered subcutaneously. 33.The method according to claim 30 wherein said polypeptide isadministered at two day intervals.
 34. The method according to claim 30wherein said polypeptide is administered at weekly intervals.
 35. Themethod according to claim 30 wherein said polypeptide is administered atbi-weekly intervals.
 36. The method according to claim 30 wherein saidpolypeptide is administered at monthly intervals.
 37. The methodaccording to claim 30 wherein said growth hormone deficiency ischildhood growth hormone deficiency.
 38. The method according to claim30 wherein said growth hormone deficiency is adult growth hormonedeficiency.